The field of the invention is diffuse reflectance spectroscopy and imaging, and in particular, the use of optical reflectance to detect epithelial pre-cancers and cancers. A promising technique under development for squamous epithelial precancer and cancer detection is optical spectroscopy. Optical techniques offer several benefits over traditional diagnostic methods that include visual inspection (through a microscope or endoscope), followed by biopsy. Light can nondestructively interact with a large number of biological molecules intrinsically present in tissues, thus providing a wealth of biochemical and structural information related to disease progression, without the need for tissue removal. Additionally, advances in sensitive detectors and optical fibers make it possible to measure optical signals rapidly and remotely from human tissues in vivo.
There are a large number of absorbers in epithelial tissues in the ultraviolet-visible (UV-VIS) spectral range. The primary absorbers within the cells in the epithelium (top layer of the epithelial tissue) are tryptophan, reduced nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FAD). The primary absorber in the underlying stroma is hemoglobin. The primary elastic scatterers in the epithelium are cellular and subcellular components including nuclei and mitochondria, while the primary elastic scatterer in the stroma is collagen.
It has been shown that the endogenous absorption and scattering contrast in precancers and early cancers of stratified squamous epithelial tissues, such as the cervix, varies with depth. Previous microscopy studies on cervical tissue slices and blocks show an increase in the contribution of NADH (source of absorption) within the epithelium, and a decrease in stromal collagen content (source of scattering) with the progression of neoplasia. Researchers have reported increased scattering in the epithelium of precancerous cervical tissues relative to that of normal tissues using confocal microscopy techniques. Moreover, there is increased blood vessel growth (source of absorption) in the stroma with cervical neoplasia, and this has been used by physicians during colposcopy to diagnose cervical precancers.
Diffuse reflectance spectroscopy provides a measure of tissue absorption as well as scattering. Based on the findings described above, depth-dependent absorption and the scattering properties (optical properties) of tissues extracted from diffuse reflectance spectra offer diagnostically useful information for the detection of epithelial precancers and early cancers.
A number of light transport models have been developed to compute optical properties from the diffuse reflectance spectrum measured from a homogeneous medium. However, since squamous epithelial tissues have a layered structure, the use of these simplistic models can cause significant errors in the extracted optical properties. Light transport models for two-layered media can overcome the intrinsic weakness of homogeneous models of light transport.
Several research groups have extended the diffusion theory to calculate the optical properties of a two-layered medium. However, diffusion theory is not valid for highly absorbing media or for small source-detector separations, as is the case in the UV-VIS. Several other research groups have proposed models based on Monte Carlo or hybrid methods. Hayakawa et al. “Peturbation Monte Carlo Methods To Solve Inverse Photon Migration Problems In Heterogeneous Tissues,” Opt. Lett. 26, 1335-1337 (2001) developed a perturbation Monte Carlo method to estimate the optical properties of a two-layered medium, in which the perturbation in photon trajectories caused by a small amount of variation in the optical properties relative to baseline values was used to guide a nonlinear optimization algorithm for the estimation of optical properties. The perturbation approach is limited in that it is constrained to small changes in the optical properties (<30% of baseline values for the scattering coefficient), and it requires that the baseline optical properties are known. Chang et al. “Analytical Model To Describe Fluorescence Spectra Of Normal And Preneoplastic Epithelial Tissue: Comparison With Monte Carlo Simulations And Clinical Measurements,” J. Biomed. Opt. 9, 511-522 (2004) proposed an analytical two-layered model to describe fluorescence spectra from epithelial tissues measured with a specific probe geometry, in which a single large fiber is used for both light excitation delivery and fluorescence emission collection. By assuming a low scattering epithelium and a highly scattering stroma, as well as one-dimensional light propagation, Beer's law was used to characterize light propagation in the epithelium while the diffusion theory was used to model light transport in the stroma. The analytical form of the model enables fast forward computation of fluorescence emission spectra. However, the presumed probe geometry and limited applicable range of tissue optical properties in each layer limits the utility of this model. Another disadvantage of previously published two-layer models in general is that they contain more free parameters compared to homogeneous models of light transport. The large number of unknowns can dramatically increase the computational time and or even cause the inversion unable to converge.
Fawzi et al. “Determination Of The Optical Properties Of A Two-Layer Tissue Model By Detecting Photons Migrating At Progressively Increasing Depths,” Appl. Opt. 42, 6398-6411 (2003) proposed an alternate sequential estimation approach for the determination of optical properties of a two-layered medium. In this approach, a flat-tip probe with a series of small source-detector separations was first used to measure spatially resolved reflectance from the top layer (thickness 5 mm), and a multivariate calibration model was used to extract the top layer optical properties. Then a flat-tip probe with a series of large source-detector separations was used to measure the phase delay and amplitude from both layers (using a frequency domain technique), and the data were fitted to a two layered frequency-domain diffusion model with the estimated top layer optical properties as known inputs. However, since this methodology is based on diffusion theory, it is not appropriate for use in the UV-VIS spectral range and or for small source-detector separations.